We propose to investigate the function of an essential malaria protease with the objective of elucidating its mechanism and potential as an antimalarial target. Termed signal peptide peptidase (SPP) due to its homology with other intramembrane proteases, the specific function of malaria SPP is unknown. SPP belongs to a family of intramembrane cleaving proteases (i-CLIPs) relevant to many disorders including Alzheimer's disease. We will use both in vivo and in vitro models of malaria to investigate its specific function at the endoplasmic reticulum (ER) where it is known to localize. We have identified the first known signal peptide substrate of PfSPP, and preliminary results indicate its protease activity is required for proper ER function. Reduction of PfSPP activity correlated with a dramatic increase in gametocytes. Chemical modulators of ER stress are known to induce gametocytogenesis. This fact, coupled to the essentiality of PfSPP, its ER localization, and its link with gametocytogenesis suggests that PfSPP may play a key role in gametocyte induction. Here we will test the hypothesis that PfSPP cleaves a conserved set of signal peptide substrates at the ER membrane required for proper ER homeostasis, and reduced PfSPP activity increases ER stress resulting in the induction of ER stress markers and gametocytogenesis. This will be tested through the following specific aims: Aim 1: Determine whether PfSPP cleaves a conserved set of substrates containing a specific amino acid motif. Aspartic acid proteases like PfSPP generally cleave a specific amino acid sequence. We will test predicted PfSPP substrates with an established cell-based transfection assay. A mutagenesis approach will be used to characterize the substrate motif required for cleavage, and design a substrate with a motif resistant to PfSPP cleavage. The mutant substrate(s) will then be used to evaluate the inhibition of parasite growth in human erythrocytes. Aim 2: Test whether reduced PfSPP activity increases the expression of ER stress markers and thus triggers gametocytogenesis. We will investigate the role of PfSPP in the regulation of ER homeostasis and stress. Using a combination of in vivo and in vitro models, we will measure the expression of known markers of ER stress and gametocytogenesis in response to reduced PfSPP activity. To measure the ER stress response, both mRNA and protein levels of known markers will be quantified using RT-PCR and immunoblotting methods, respectively. Furthermore, SPP inhibitors will be evaluated in vivo for their use as potential antimalarial therapeutics.